The structure of tissues (such as heart or skeletal muscle) undergoes substantial remodeling in disease, including macroscopic changes, e.g.: overall geometry;vascular network reorganization;fiber and sheet orientation;and interstitial fibrosis;and microscopic changes, e.g.: redistribution of molecular expression;and the energetic state of mitochondria. There is an urgent need to understand the nature of this remodeling, because all of these processes impact on the function of these tissues in health and disease, but existing methods for tissue reconstruction are low-resolution, while current high-resolution sub- cellular imaging cannot be expanded to tissues and whole organs. Here we describe an unprecedented approach by which two-photon microscopy will be used to perform whole-tissue 3D reconstructions at micron- level spatial resolution, bridging the gap from micro to macro scale. We request a state-of-the-art Zeiss 7MP dedicated multiphoton microscope system for the multiphoton- excited fluorescence imaging and automated 3D reconstruction of tissues, to be used by multiple NIH-funded investigators to accomplish research not currently possible with existing equipment. Integration of a motorized stage and automated microtome allow this instrument to produce images at a higher resolution, for a larger volume of tissue, than has been yet achieved, making three-dimensional reconstructions of large-tissue- volume images possible. The system will initially have 7 major users and will be fully utilized for long imaging sessions of large tissues;as research evolves, minor users may be added. This will be the only instrument of its kind at the Johns Hopkins University. All other multiphoton microscopes at the university are used for different applications and cannot be used or modified for our purposes;this is the only one at JHU to be used for these time-consuming tissue reconstructions. The University has identified this need and backs our proposal with financial support for infrastructure to accommodate this imaging system. Relevance: The proposed system removes roadblocks to a wide range of currently NIH-funded research, and opens the door to a breadth of future work of significant impact on human health. Applications include: atlases of healthy and diseased hearts in multiple species;and computational analysis of both the population-level and individual-level anatomical changes that occur. Many of the downstream applications involve using the high- resolution datasets generated to create complex anatomically-detailed and individualized computational models of cardiac electrophysiology, electromechanics, blood flow and drug delivery, to simulate Sudden Cardiac Death, Cardiac Defibrillation, Peripheral Arterial Disease and more. In fact, we envision most data acquired being used at least three times: as an image;as a component of an atlas;and as the basis of at least one computational model.